title: Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay.
abstract: BACKGROUND
  Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts.
  
  
  METHODS
  The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF).
  
  
  RESULTS
  MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis.
  
  
  CONCLUSIONS
  In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential.
authors.affiliation: Louvain centre for Toxicology and Applied Pharmacology, Université catholique de Louvain, Avenue E, Mounier, 52 - bte B1,52,12, 1200 Brussels, Belgium. giulia.vietti@uclouvain.be.
authors.forename: Giulia
authors.initials: G
authors.lastname: Vietti
authors.forename: Saloua
authors.initials: S
authors.lastname: Ibouraadaten
authors.forename: Mihaly
authors.initials: M
authors.lastname: Palmai-Pallag
authors.forename: Yousof
authors.initials: Y
authors.lastname: Yakoub
authors.forename: Christian
authors.initials: C
authors.lastname: Bailly
authors.forename: Ivana
authors.initials: I
authors.lastname: Fenoglio
authors.forename: Etienne
authors.initials: E
authors.lastname: Marbaix
authors.forename: Dominique
authors.initials: D
authors.lastname: Lison
authors.forename: Sybille
authors.initials: S
authors.lastname: van den Brule
mesh_terms: D000818:Animals; D017638:Asbestos, Crocidolite / Q000737:chemistry / Q000633:toxicity; D041702:BALB 3T3 Cells; D001681:Biological Assay; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008855:Microscopy, Electron, Scanning; D037742:Nanotubes, Carbon / Q000737:chemistry / Q000633:toxicity*; D010316:Particle Size; D011237:Predictive Value of Tests; D011658:Pulmonary Fibrosis / Q000139:chemically induced* / Q000473:pathology; D015203:Reproducibility of Results; D013499:Surface Properties
chemical_list: D037742:Nanotubes, Carbon; D017638:Asbestos, Crocidolite